Molecular detection of tick-borne bacteria and protozoa in cervids and wild boars from Portugal

Background: Wildlife can act as reservoir of different tick-borne pathogens, such as bacteria, parasites and viruses. The aim of the present study was to assess the presence of tick-borne bacteria and protozoa with veterinary and zoonotic importance in cervids and wild boars from the Centre and South of Portugal.Methods: One hundred and forty one blood samples from free-ranging ungulates including 73 red deer (Cervus elaphus), 65 wild boars (Sus scrofa) and three fallow deer (Dama dama) were tested for the presence of Anaplasma marginale/A. ovis, A. phagocytophilum, Anaplasma/Ehrlichia spp., Babesia/Theileria spp., Borrelia burgdorferi (sensu lato) (s.l.), and Rickettsia spp. DNA by PCR.Results: Anaplasma spp. DNA was detected in 33 (43.4 %) cervids (31 red deer and two fallow deer) and in two (3.1 %) wild boars while Theileria spp. were found in 34 (44.7 %) cervids (32 red deer and two fallow deer) and in three (4.6 %) wild boar blood samples. Sequence analysis of msp4 sequences identified A. marginale, A. ovis, while the analysis of rDNA sequence data disclosed the presence of A. platys and A. phagocytophilum and T. capreoli and Theileria sp. OT3. Anaplasma spp./Theileria spp. mixed infections were found in 17 cervids (22.4 %) and in two wild boars (3.1 %). All samples were negative for Babesia sp., B. burgdorferi (s.l.), Ehrlichia sp. or Rickettsia sp.Conclusions: This is the first detection of Anaplasma marginale, A. ovis, A. phagocytophilum, A. platys, Theileria capreoli and Theileria sp. OT3 in cervids and wild boars from Portugal. Further studies concerning the potential pathogenicity of the different species of Anaplasma and Theileria infecting wild ungulates, the identification of their vector range, and their putative infectivity to domestic livestock and humans should be undertaken

Contributions to the phylogeny of Ixodes (Pholeoixodes) canisuga, I. (Ph.) kaiseri, I. (Ph.) hexagonus and a simple pictorial key for the identification of their females

Background: In Europe, hard ticks of the subgenus Pholeoixodes (Ixodidae: Ixodes) are usually associated with burrow-dwelling mammals and terrestrial birds. Reports of Pholeoixodes spp. from carnivores are frequently contradictory, and their identification is not based on key diagnostic characters. Therefore, the aims of the present study were to identify ticks collected from dogs, foxes and badgers in several European countries, and to reassess their systematic status with molecular analyses using two mitochondrial markers. Results: Between 2003 and 2017, 144 Pholeoixodes spp. ticks were collected in nine European countries. From accurate descriptions and comparison with type-materials, a simple illustrated identification key was compiled for adult females, by focusing on the shape of the anterior surface of basis capituli. Based on this key, 71 female ticks were identified as I. canisuga, 21 as I. kaiseri and 21 as I. hexagonus. DNA was extracted from these 113 female ticks, and from further 31 specimens. Fragments of two mitochondrial genes, cox1 (cytochrome c oxidase subunit 1) and 16S rRNA, were amplified and sequenced. Ixodes kaiseri had nine unique cox1 haplotypes, which showed 99.2-100% sequence identity, whereas I. canisuga and I. hexagonus had eleven and five cox1 haplotypes, respectively, with 99.5-100% sequence identity. The distribution of cox1 haplotypes reflected a geographical pattern. Pholeoixodes spp. ticks had fewer 16S rRNA haplotypes, with a lower degree of intraspecific divergence (99.5-100% sequence identity) and no geographical clustering. Phylogenetic analyses were in agreement with morphology: I. kaiseri and I. hexagonus (with the similar shape of the anterior surface of basis capituli) were genetically more closely related to each other than to I. canisuga. Phylogenetic analyses also showed that the subgenus Eschatocephalus (bat ticks) clustered within the subgenus Pholeoixodes. Conclusions: A simple, illustrated identification key is provided for female Pholeoixodes ticks of carnivores (including I. hexagonus and I. rugicollis) to prevent future misidentification of these species. It is also shown that I. kaiseri is more widespread in Europe than previously thought. Phylogenetic analyses suggest that the subgenus Pholeoixodes is not monophyletic: either the subgenus Eschatocephalus should be included in Pholeoixodes, or the latter subgenus should be divided, which is a task for future studies

Significance of red deer [Cervus elaphus] in the ecology of Borrelia burgdorferi sensu lato

Background. Red deer (Cervus elaphus) is one of the most important host of the adult tick (Ixodes ricinus) which is the basic vector of the Lyme disease causative agent — Borrelia burgdorferi sensu lato in Europe. The aim of the present study was to establish the role of red deer in the transmission of B. burgdorferi s.l. Material and methods. Tissues from 74 red deers were evaluated and the presence of B. burgdorferi s.l DNA was identified using nested PCR technique based on fla gene. The identification of species belonging to B. burgdorferi s.l complex was performed after restriction digestion of nested PCR product with DdeI enzyme and sequencing of nested PCR product. The study included also 55 isolates of I. ricinus females removed from red deer and 466 ticks (73 adult and 393 nymphs) collected from the vegetation in the area where the red deer lives. Results. There were no DNA of B. burgdorferi s.l complex in the red deer tissues and in ticks removed from deer, however in one tick removed from deer the DNA of other Borrelia species — B. miyamotoi was identified. In ticks collected from vegetation 3 species belonging to B. burgdorferi s.l. complex were identified: B. garinii (3.2% ticks studied), B. afzelii (6.9%) and B. valaisiana (3.6%), however DNA of B. miyamotoi was absent. These results confirm inability of survival of B. burgdorferi s.l. species in tick I. ricinus feeding on red deer blood. However there is a possibility of survival of B. miyamotoi in presence of deer blood at least in ticks feeding on red deer. The main role of red deer in keeping the constant infection level of B. burgdorferi s.l. in the whole population of I. ricinus ticks does not concern B. miyamotoi

Wystepowanie Ixodes ricinus na wybranych terenach rekreacyjnych b. wojewodztwa szczecinskiego. Cz.2

The aim of the study was to estimate the occurrence (the quantative and rate percent) of Ixodes ricinus in the popular recreation areas in Szczecin (Arkonka, Osów, Głębokie, Landscape Park of Szczecin, Dąbie Forest Park, Zdroje Forest Park) and in province of Szczecin (Forest of Goleniów, Ińsko, Pobierowo, Chojna). Investigations were performed in 1998 year, twice on each places; in May/June and repeted in September/October. The temperature and humidity of air were measured. Obtained specimens were regard of sex and growing stage during each collection. A total of 2.055 specimens collected 49% were nymphs, 13.9% female, 11.3% male and 25.8% larvaes. The nymphs the most frequently were in spring when humidity of air was 55% and temperature 24°C. The larvaes, in autumn were most frequently (31.4%) then in spring (20.5%) when the temperature of the air was 18-22°C, and the humidity from 60 to 85% during the collections

OCCURRENCE OF SPIROCHETES BORRELIA BURGDORFERI S. L. IN TICKS IXODES RICINUS IN THE FORESTS OF SZCZECIN PROVINCE

Presently acquired results are the outcome of, as for today, pilot-scale, preliminary study, although they were based on a tick (Ixodes ricinus) population of 533 individuals, collected from the vicinity of Szczecin. The areas sampled have been considered as recreational, which means they are frequented by a large number of people. The ticks were collected in 1996 in two seasons: spring-summer and summer-autumn. The overall prevalence of the spirochetes B. burgdorferi sensu lato, in the tick population studied, was 12.6%, while that of the spring-summer season reached 25.5%, decreasing in the summer-autumn season down to 2.3%

Wystepowanie Ixodes ricinus na wybranych terenach rekreacyjnych b. wojewodztwa szczecinskiego. Cz.3

The occurrence of Ixodes ricinus in the selected recreative areas in the province of Szczecin. Part III. Within the last years, the incidences of diseascs transmitted by Ixodes ricinus tick have rapidly increased. We estimated the occurrence of Ixodes ricinus in the popuJlr recreation urban areas in Szczecin and in the Province of Szczecin. The study was carried out in 1999, with two samples at each site, and were compared with data of 1998. The temperature and humidity of air were measured. The most ticks were found in the range of 70-80% relative humidity of air. Among 3.198 specimens collected 59.5% were nymphs, 19.0% larvae, 11.1 % females and 10.4% males. The nymphs were the most frequent in spring and in autumn, while the larvae were most frequent in autumn (26.2%) then in spring (11.9%)